It is generally recognized that reduced delivery of oxides of nitrogen in the smoke of tobacco products is desirable. Therefore, a number of methods have been developed to reduce the levels of nitrogen oxide precursors, such as nitrates, in smoking products. Those prior art methods are of three main types--ion exchange, crystallization and microbiological.
Ion exchange-based methods for reducing the levels of nitrate in tobacco materials are described, for example, in U.S. Pat. Nos. 3,616,801, 3,847,164 and 4,253,929. These methods, such as ion exchange, ion retardation and electrodialysis, while perhaps feasible on a small scale, are both expensive and impractical on a larger scale. In addition, regeneration of the required resins and membranes, isolation and disposal of the nitrogen-containing by-products and cost and disposal of the spent resins and membranes add to the cost of the processes.
Crystallization-based methods for reducing nitrate concentration in tobacco materials are described, for example, in U.S. Pat. No. 4,131,118. These methods are usable in large scale processes and permit the rapid isolation of the nitrogen-containing by-products. However, these methods are not only limited by the necessity to dispose of the by-product, they are limited by the level of nitrate-nitrogen reduction that can be obtained in them. For example, tobacco extracts after treatment by these processes usually contain between about 0.4% to 0.45% (4000-4500 ppm) nitrate-nitrogen. Further reductions in the nitrate-nitrogen concentration of these extracts would plainly be advantageous, if they could be obtained in a cost effective manner.
A wide variety of microbial processes and microorganisms useful in those processes have also been proposed for reducing the levels of certain nitrogen-containing compounds in tobacco materials. These processes and organisms, which may be either aerobic or anaerobic, make use of both dissimilatory and assimilatory pathways to metabolize the nitrogen-containing compounds. These processes and organisms, for example, include those of U.S. Pat. No. 3,747,608, British patent specification No. 1,557,253 (stated to be based on U.S. application Ser. No. 883,449, filed Mar. 6, 1978, now U.S. Pat. No. 4,308,877), UK patent specification Nos. 2,014,031 (based on Luxembourg application No. 79039, filed Feb. 9, 1978, now Luxembourg patent No. 79039), 2,023,995 (stated to be based on U.S. application Ser. No. 916,322, filed June 15, 1978) and 2,028,628 (stated to be based on U.S. application Ser. No. 916,323, filed June 15, 1978), Canadian patent No. 1,081,076 (based on Luxembourg application No. 77272, filed May 6, 1977, and Luxembourg patent No. 77272), now Luxembourg application No. 77872, filed July 29, 1977, now Luxembourg patent No. 77872), European patent No. 5,082 (based on U.S. application Ser. No. 900,044, filed Apr. 25, 1978) and West German patent application No. P3100715.5, filed Jan. 13, 1981 (Offenlegungsschriften No. DE 3100715).
While some of these processes make use of bacteria that belong to the indigenous microflora of tobacco, each employs only non-thermophilic microorganisms as the active microbial agent. Each also employs only low temperature fermentation conditions--5.degree.-40.degree. C. For example, British patent specification No. 1,557,253 employs 5.degree.-35.degree. C. Canadian patent No. 1,081,076--25.degree.-35.degree. C., UK patent specification No. 2,014,031--25.degree.-35.degree. C., UK patent application specification No. 2,023,995--20.degree.-40.degree. C., UK patent application specification No. 2,028,628--5.degree.-37.degree. C., European patent No. 5,082--30.degree.-40.degree. C., West German patent application (Offenlegungsschriften No. DE 3100715).--30.degree. C. and U.S. Pat. No. 3,747,608--24.degree.-40.degree. C.
Most of these processes also require that the tobacco materials be terminally sterilized (e.g., 121.degree. C. for 15 min at 15 psig) before contact with the microorganisms and that the fermentation be conducted under substantially aseptic conditions. The various anaerobic processes also usually require sparging of the fermentation broth with inert gases or other treatments to limit the oxygen concentration.
A number of these processes also require various additives to be incorporated into the fermentation broths or to supplement the tobacco material isolated from those broths after fermentation. For example, British patent specification No. 1,557,253 requires various organic compounds to be added to the tobacco materials, Canadian patent No. 1,081,076 and UK patent application No. 2,014,031A require D-glucose and other additives and West German patent application No. P3100715.5 requires that sugars be added to the broth. Plainly, any requirement for such additives increases the cost of such processes and may result in non-tobacco compounds being incorporated into the tobacco materials.
Other microbial-based processes for treating tobacco are also known in the art. For example, U.S. Pat. Nos. 2,000,855, 3,747,608 and 4,037,609 purport to describe microbial processes and microorganisms for degrading nicotine that may be present in tobacco. These processes, although again perhaps making use of bacteria that belong to the indigenous microflora of tobacco, are also non-thermophilic and employ low temperature fermentation conditions. E.g., 24.degree.-40.degree. C. (U.S. Pat. No. 3,747,608), 20.degree.-45.degree. C. (U.S. Pat. No. 4,037,609) and 30.degree.-40.degree. C. (U.S. Pat. No. 2,000,855).
In addition, Japanese patent No. 73 49,999 (C.A. 79:123942x), S. A. Ghabrial, "Studies On The Microflora Of Air-Cured Burley Tobacco", Tobacco Science, pp. 80-82 (1976), W. O. Atkinson et al., Ky. Agr. Exp. Sta. Lexington Ann. Report, 86, p. 22 (1973), A. Koiwai et al., Tob. Sci, 15, pp. 41-3 (1971) and U.S. Pat. No. 2,317,792 purport to describe other microbial-based fermentation and curing processes for tobacco. Again, each of these processes employs non-thermophilic organisms and low temperature fermentation conditions, e.g., 25.degree.-50.degree. C. (Japanese patent No. 73 49,999), 30.degree.-35.degree. C. (S. A. Ghabrial) and 30.degree.-40.degree. C. (A. Koiwai et al.).
Biological processes for reducing the concentration of nitrogen-containing compounds in waste water are also known in the art. These include, for example, U.S. Pat. Nos. 3,829,377 and 4,225,430. Again, they employ non-thermophilic microorganisms and low temperature conditions, e.g., 10.degree.-50.degree. C. (U.S. Pat. No. 3,829,377). Again, they require a carbon source to be added to the waste water, e.g., molasses (U.S. Pat. No. 4,225,430) and C.sub.1 to C.sub.3 hydrocarbons (U.S. Pat. No. 3,829,377).
Finally, the growth of thermophilic microorganisms on "sweating" tobacco is known to occur. However, such organisms have not been employed to reduce the content of nitrogen-containing compounds in tobacco. Rather, they have only been described to affect the aroma and mildness of cigar tobacco. Such processes include, for example, those of C. F. English et al., "Isolation of Thermophiles From Broadleaf Tobacco And Effect Of Pure Culture Inoculation On Cigar Aroma And Mildness", Applied Microbiol., 15, pp. 117-19 (January 1967) and B. Dumery and J. P. Albo, "Participation of Microorganisims In The Fermentation Of Dark Tobacco Submitted To A "Pre-Storage-Thermic Treatment Storage" Type Of Process", A du Tabac, Sect. 2-16, Bergerac, S.E.I.T.A. (1979-80).
Microorganisms are also known to denitrify soil and sewage. Such processes are described, for example, in M. Henze Christensen and P. Harremoes, "Biological Denitrification of Sewage: A Literature Review", Prog. Wat. Tech., 8, pp. 509-55 (1977); D. D. Focht, "The Effect Of Temperature, pH And Aeration On The Production Of Nitrous Oxide And Gaseous Nitrogen--A Zero-Order Kinetic Model," Soil Science, 118, pp. 173-79 (1974); J. M. Bremner and K. Shaw, "Denitrification In Soil II. Factors Affecting Denitrification", J. Agricultural Science, 51, pp. 40-52 (1958); and H. Nommik, "Investigations On Denitrification In Soil", Acta Agriculture Scandinavica, 6, pp. 195-228 (1956). None of these references discloses the use of thermophilic organisms in denitrification. Moreover, the ones that report that the rate of nitrate reduction increases with increasing fermentation temperatures attribute the observed rate increase to the standard temperature effect on a biochemical reaction, not the activation and growth of a new class of microorganisms. And, none suggests such temperature-dependent rate increases would be observed in tobacco fermentation.
Therefore, none of these prior processes makes use of high temperature processes and thermophilic microorgansims to reduce the content of nitrogen-containing compounds in tobacco materials. Neither do any of these prior processes suggest that these nitrogen-containing compounds of tobacco materials could be metabolized at high temperatures via dissimilatory pathways by thermophilic microorganisms or that such organisms might be isolated from the indigenous microflora of tobacco. Neither do these prior processes suggest that such dissimilatory metabolism could occur in the absence of additives to the fermentation broth or tobacco or under substantially non-aseptic fermentation conditions.